Method for producing biomolecule diagnostic devices

ABSTRACT

A biosensor includes a substrate member with a pattern of active areas of receptive material and a pattern of blocking material layers. The receptive material and blocking material are attached to the substrate member with a photo-reactive crosslinking agent activated in a masking process. The receptive material is specific for an analyte of interest.

PRIORITY INFORMATION

The present application claim priority to and is a divisionalapplication of U.S. application Ser. No. 10/139,025 filed on May 3, 2002of Cohen, et al., now U.S. Pat. No. 7,771,922, which is incorporated byreference herein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to the field of detectinganalytes in a medium, and more particularly to a process for preparinganalyte-specific diagnostic sensors to indicate the presence of theanalyte in a medium in, for example, a diffraction/holography format.

BACKGROUND

There are many systems and devices available for detecting a widevariety of analytes in various media. Many of the prior systems anddevices are, however, relatively expensive and require a trainedtechnician to perform the test. A need has been recognized in the artfor biosensor systems that are easy and inexpensive to manufacture, andcapable of reliable and sensitive detection of analytes. Reference ismade, for example, to U.S. Pat. Nos. 5,922,550; 6,060,256; and 6,221,579B1.

Various advances have been made in the industry for producingbiosensors. For example, U.S. Pat. No. 5,512,131 to Kumar, et al.,describes a device that includes a polymer substrate having a metalcoating. An analyte specific receptor layer is stamped onto the coatedsubstrate. A diffraction pattern is generated when an analyte binds tothe device. A visualization device, such as a spectrometer, is then usedto determine the presence of the diffraction pattern. A drawback to thistype of device is, however, the fact that the diffraction pattern is notdiscernible by the naked eye and, thus, a complex visualization deviceis needed to view the diffraction pattern. Also, the device is generallynot able to detect smaller analytes that do not produce a noticeablediffraction pattern.

U.S. Pat. No. 5,482,830 to Bogart, et al., describes a device thatincludes a substrate which has an optically active surface exhibiting afirst color in response to light impinging thereon. This first color isdefined as a spectral distribution of the emanating light. The substratealso exhibits a second color which is different from the first color.The second color is exhibited in response to the same light when theanalyte is present on the surface. The change from one color to anothercan be measured either by use of an instrument, or by the naked eye. Adrawback with the device is, however, the relatively high cost of thedevice and problems associated with controlling the various layers thatare placed on the wafer substrate.

Contact printing techniques have been explored for producing biosensorshaving a self-assembling monolayer. U.S. Pat. No. 5,922,550 describes abiosensor having a metalized film upon which is printed (contactprinted) a specific predetermined pattern of an analyte-specificreceptor. The receptor materials are bound to the self-assemblingmonolayer and are specific for a particular analyte or class ofanalytes. Attachment of a target analyte that is capable of scatteringlight to select areas of the metalized plastic film upon which thereceptor is printed causes diffraction of transmitted and/or reflectedlight. A diffraction image is produced that can be easily seen with theeye or, optionally, with a sensing device. U.S. Pat. No. 6,060,256describes a similar device having a metalized film upon which is printeda specific predetermined pattern of analyte-specific receptor. The '256patent is not limited to self-assembling monolayers, but teaches thatany receptor which can be chemically coupled to a surface can be used.The invention of the '256 patent uses methods of contact printing ofpatterned monolayers utilizing derivatives of binders formicroorganisms. One example of such a derivative is a thiol. The desiredbinding agent can be thiolated antibodies or antibody fragments,proteins, nucleic acids, sugars, carbohydrates, or any otherfunctionality capable of binding an analyte. The derivatives arechemically bonded to metal surfaces such as metalized polymer films, forexample via a thiol.

A potential issue of the contact printing techniques described above forproducing diffraction-based biosensors is the possibility ofcontamination from the print surface (i.e., stamp) during the printingprocess. Also, there is the possibility of uneven application or inkingof the substances due to pressure and contact variations inherent in theprocess, as well as surface energy variations.

The present invention relates to a biosensor system that is easy andinexpensive to manufacture, is capable of reliable and sensitivedetection of analytes, and avoids possible drawbacks of conventionalmicrocontact printing techniques.

SUMMARY OF THE INVENTION

Objects and advantages of the invention will be set forth in part in thefollowing description, or may be obvious from the description, or may belearned through practice of the invention.

The present invention provides a relatively inexpensive yet sensitivebiosensor device, a method for producing such biosensor devices, and amethod for detecting analytes of interest present in a medium.

The biosensor includes a substrate upon which a layer containing abifunctional crosslinking agent is applied in a light protectedenvironment generally uniformly over an entire surface of the substratemember. This agent has a molecular make-up such that one side of themolecule reacts with and forms a relatively strong bond with thesubstrate member. The other end of the agent has a photo-reactive groupsuch that, in the presence of or after exposure to an irradiating energysource of sufficient amplitude and frequency, the agent cross-links withany other molecules in close proximity. Examples of such bifunctionalcrosslinking agents include SANPAH (N-Succinimidyl2-[p-azido-salicylamido]ethyl-1,3′-dithiiopropionate); SAND(Sulfosuccinimidyl2-[m-azido-o-nitro-benzamido]ethyl-1,3′-dithiopropionate); and ANB-NOS(N-5-Azido-2-nitrobenzoyloxysuccinimide).

The substrate may be any one of a wide variety of suitable materials,including plastics, metal coated plastics and glass, functionalizedplastics and glass, silicon wafers, foils, glass, etc. Desirably, thesubstrate is flexible, such as a polymeric film, in order to facilitatethe manufacturing process.

The crosslinking agent layer is desirably applied in a light-protectedenvironment by any number of known techniques, including dipping,spraying, rolling, spin coating and any other technique wherein thelayer can be applied generally uniformly over the entire test surface ofthe substrate. The invention also includes contact printing methods ofapplying the photo-reactive crosslinking agent layer.

In one embodiment, a layer containing a receptive material (e.g.,biomolecules) is then applied over the photo-reactive crosslinkingagent, desirably in a light protected environment. The receptivematerial is selected so as to have a particular affinity for an analyteof interest. The receptive material layer may be applied by any numberof known techniques, including dipping, spraying, rolling, spin coatingand any other technique wherein the layer can be applied generallyuniformly over the entire test surface of the substrate. The inventionalso includes contact printing methods of applying the receptivematerial layer, as long as such methods are conducted in a manner toprevent inconsistent inking and contamination from contact during theinitial coating process.

A mask having a pattern of exposed and shielded areas is placed over thesubstrate. The mask may include any desired pattern of protected orshielded areas and exposed areas (e.g., transparent or translucentareas, as well as holes or openings in the mask structure). The exposedareas of the mask will correspond to a pattern of active receptivematerial areas (biomolecules) and the shielded areas of the mask willdefine a pattern of inactive blocking material areas. The mask andsubstrate combination is then irradiated with an energy sourcesufficient to activate the crosslinking agent such that thephoto-reactive groups crosslink or attach to the biomolecules in theexposed areas of the mask and thus “secure” the biomolecules relative tothe substrate in the exposed areas.

In a light protected environment, the mask is removed and the unattachedbiomolecules that were underlying the shielded areas of the mask areremoved from the substrate member by, for example, a washing or rinsingprocedure. The crosslinked biomolecules remain attached to the substratemember in a pattern corresponding to the exposed areas of the mask.

In one embodiment, desirably in a light protected environment, amaterial containing molecules different than the biomolecules selectedfor the analyte of interest is applied to the substrate member. These“different” molecules will serve, in essence, to fill in or block theregions on the substrate between the active receptive material areas andmay be, for example, biomolecules that specifically do not have anaffinity for the analyte of interest. In general, any type of blockingmolecule may be used for this purpose.

The substrate member is then exposed again to the energy source for asufficient time to activate the photo-reactive groups of thecrosslinking agent. The blocking molecules become crosslinked orattached to the substrate member in a pattern corresponding to theoriginally shielded areas of the mask.

The substrate member is finally washed or rinsed to remove any remainingunattached blocking molecules. A patterned monolayer of defined areas ofactive biomolecules for an analyte of interest interposed between areasof blocking molecules remains on the substrate.

In an alternative embodiment, the blocking material may be first appliedto the crosslinking agent and exposed through a mask. The receptivematerial would then be applied after the masking process. Thus, in thisembodiment, the pattern of active receptive material areas correspondsto the shielded areas of the mask, and the pattern of inactive blockingmaterial areas corresponds to the exposed areas of the mask.

It should be appreciated that the invention is not limited to anyparticular pattern defined by the mask. Virtually any number andcombination of exposed shapes or openings are possible. In oneparticular embodiment, the pattern is defined by about 10 microndiameter pixels at a spacing of about 5 microns over the test surface ofthe substrate.

The photo-reactive crosslinking agent is irradiated with an energysource selected particularly for activating the specific type ofphoto-reactive agent. The invention is not limited to any particularenergy source. For example, the energy source may be a light source,e.g., an ultraviolet (UV) light source, an electron beam, a radiationsource, etc. Care should be taken such that the exposure is sufficientfor activating the crosslinking agent, but does not destroy or breakdown the receptive material or does not damage the underlying substratemember. Upon subsequent exposure of the biosensor to a medium containingan analyte of interest, the analyte binds to the biomolecules in theactive areas. The biosensor will then diffract transmitted light in adiffraction pattern corresponding to the active areas. The diffractionpattern may be visible to the naked eye or, optionally, viewed with asensing device.

In the case where an analyte does not scatter visible light because theanalyte is too small or does not have an appreciable refractive indexdifference compared to the surrounding medium, a diffraction-enhancingelement, such as polymer microparticles, may be used. Thesemicorparticles are coated with a binder or receptive material that alsospecifically binds to the analyte. Upon subsequent coupling of theanalyte to both the patterned biomolecules in the receptive materiallayer as well as the microparticles, a diffraction image is producedwhich can be easily seen with the eye or, optionally, with a sensingdevice.

By “diffraction” it is meant the phenomenon, observed when waves areobstructed by obstacles, of the disturbance spreading beyond the limitsof the geometrical shadow of the object. The effect is marked when thesize of the object is of the same order as the wavelength of the waves.In the present invention, the obstacles are analytes (with or without orattached microparticles) and the waves are light waves.

In another embodiment of the present invention, nutrients or receptorsfor a specific class of microorganisms can be incorporated as thereceptive material in the active areas. In this way, very lowconcentrations of microorganisms can be detected by first contacting thebiosensor of the present invention with the nutrients incorporatedtherein and then incubating the biosensor under conditions appropriatefor the growth of the bound microorganism. The microorganism is allowedto grow until there are enough organisms to form a diffraction pattern.

The present invention provides a low-cost, disposable biosensor that canbe mass produced. The biosensors of the present invention can beproduced as a single test for detecting an analyte or it can beformatted as a multiple test device. The uses for the biosensors of thepresent invention include, but are not limited to, detection of chemicalor biological contamination in garments, such as diapers, the detectionof contamination by microorganisms in prepacked foods such as meats,fruit juices or other beverages, and the use of the biosensors of thepresent invention in health diagnostic applications such as diagnostickits for the detection of proteins, hormones, antigens, nucleic acids,microorganisms, and blood constituents. It should be appreciated thatthe present invention is not limited to any particular use orapplication.

These and other features and advantages of the present invention willbecome apparent after a review of the following detailed description ofthe disclosed embodiments.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a method for producingbiosensors according to the invention by a masking process.

DETAILED DESCRIPTION

The invention will now be described in detail with reference toparticular embodiments thereof. The embodiments are provided by way ofexplanation of the invention, and not meant as a limitation of theinvention. For example, features described or illustrated as part of oneembodiment may be used with another embodiment to yield still a furtherembodiment. It is intended that the present invention include these andother modifications and variations as come within the scope and spiritof the invention.

The present invention features improved biosensing devices, and methodsfor using such biosensing devices, for detecting and quantifying thepresence or amount of an analyte of interest within a medium. Theanalytes that can be detected by the present invention include, but arenot limited to, microorganisms such as bacteria, yeasts, fungi, viruses,proteins, nucleic acids, and small molecules. The biosensing devicesaccording to the invention are relatively inexpensive and haveadvantages over conventional micro-contact printed biosensors.

The present invention comprises, in broad terms, a process of definingan active pattern of analyte-specific receptive material on a substratesurface by photo-masking the substrate. A layer containing aphoto-reactive crosslinking agent is first applied to a surface of thesubstrate member. The molecular structure of the crosslinking agent issuch that one functional group or side thereof reacts with or attachesto the surface of the substrate member. Another functional group or sideof the crosslinking agent is photoactivatable such that, in the presenceof the correct amplitude and frequency of electromagnetic radiation, thegroup will cross-link with other molecules in close proximity.Specifically, the photo-reactive crosslinking agent may be any one of anumber of substances, including SANPAH (N-Succinimidyl2-[p-azido-salicylamido]ethyl-1,3′-dithiiopropionate); SAND(Sulfosuccinimidyl2-[m-azido-o-nitro-benzamido]ethyl-1,3′-dithiopropionate); and ANB-NOS(N-5-Azido-2-nitrobenzoyloxysuccinimide).

A generally uniform coating of the receptive material or the blockingmaterial is then applied to the substrate surface over the crosslinkingagent layer. A mask is placed over the substrate, and the mask andsubstrate combination is irradiated with an energy source specificallyselected to activate the photo-reactive group of the crosslinking agent.In its basic form, the “mask” serves to shield at least one area orsection of the substrate member from the irradiating energy source andto expose at least one adjacent section to the energy source. Forexample, the mask may be a generally transparent or translucent blank(e.g., a strip of material) having any pattern of shielded regionsprinted or otherwise defined thereon. The exposed unshielded regions ofthe mask correspond to the exposed areas of the substrate member.Alternatively, the mask may simply be a single object placed upon thesubstrate. The area under the object would be shielded and thus definean active area of the receptive material, and the area around the objectwould be exposed to the energy source and thus define an area ofinactive receptive material. Alternatively, the object may have anypattern of openings defined therethrough corresponding to the exposedareas.

As mentioned, the energy source is selected so that the reactive groupof the exposed crosslinking agent is activated and thus attaches orcrosslinks with the overlying material (the receptive material orblocking material). The photo-reactive agent in the regions shielded bythe mask remains “unactivated.” Thus, upon removal of the mask, apattern of either active receptive material or blocking material isdefined on the substrate member corresponding to the pattern of theexposed areas of the mask. It should be understood that “pattern”includes as few as one active area and one inactive area.

The receptive material or blocking material that was under the shieldedareas of the mask (and thus not crosslinked with the crosslinking agent)is removed from the substrate in any suitable cleansing process, such asrinsing the substrate with water or a buffer solution.

A generally uniform layer of the respective other material is thenapplied to the substrate member. For example, if the receptive materialwas applied to the substrate before the masking process, the blockingmaterial is subsequently applied. Likewise, if the blocking material wasfirst applied, the receptive material is subsequently applied. Thesubstrate member is then exposed to the energy source a second time soas to activate the remaining crosslinking agent in the areas of thesubstrate member that were shielded by the mask in the masking process.Exposure is sufficient to activate the agent without damaging ordeactivating the receptive material (if the receptive material wasapplied first). During or after exposure to the energy source, theremaining crosslinking agent is activated and, thus, a pattern of theother material (blocking material or receptive material) is defined onsubstrate member corresponding to the pattern of the shielded areas ofthe mask. Any remaining un-linked material is then cleaned from thesubstrate member in an appropriate cleaning process, e.g., a rinsingstep.

Upon subsequent exposure of the biosensor to a medium containing theanalyte of interest, such analyte will bind to the biomolecules in theactive receptive material areas. The analyte results in diffraction oftransmitted and/or reflected light in a visible diffraction patterncorresponding to the active receptive material areas. As discussed ingreater detail below, an enhancer may be used for enhancing diffractionfrom extremely small analytes.

The analytes that are contemplated as being detected using the presentinvention include, but are not limited to, bacteria; yeasts; fungi;viruses; rheumatoid factor; antibodies, including, but not limited toIgG, IgM, IgA, IgD, and IgE antibodies; carcinoembryonic antigen;streptococcus Group A antigen; viral antigens; antigens associated withautoimmune disease; PSA (prostate specific antigen) and CRP (C-reactiveprotein) antigens; allergens; tumor antigens; streptococcus Group Bantigen; HIV I or HIV II antigen; or host response (antibodies) to theseand other viruses; antigens specific to RSV or host response(antibodies) to the virus; antigen; enzyme; hormone; polysaccharide;protein; lipid; carbohydrate; drug or nucleic acid; Salmonella species;Candida species, including, but not limited to Candida albicans andCandida tropicalis; Neisseria meningitides groups A, B, C, Y and W sub135, Streptococcus pneumoniae; E. coli; Haemophilus influenza type A/B;an antigen derived from microorganisms; a hapten; a drug of abuse; atherapeutic drug; an environmental agent; and antigens specific toHepatitis. In broad terms, the “analyte of interest” may be thought ofas any agent whose presence or absence from a biological sample isindicative of a particular health state or condition.

It is also contemplated that nutrients for a specific class ofmicroorganism can be incorporated into the receptive material layer. Inthis way, very low concentrations of microorganisms can be detected byexposing the biosensor of the present invention with the nutrientsincorporated therein to the suspect medium and then incubating thebiosensor under conditions appropriate for the growth of the boundmicroorganism. The microorganisms are allowed to grow until there areenough organisms to form a diffraction pattern. Of course, in somecases, the microorganism is present or can multiply enough to form adiffraction pattern without the presence of a nutrient in the activereceptive material areas.

The receptive material is characterized by an ability to specificallybind the analyte or analytes of interest. The variety of materials thatcan be used as receptive material is limited only by the types ofmaterial which will combine selectively (with respect to any chosensample) with a secondary partner. Subclasses of materials which fall inthe overall class of receptive materials include toxins, antibodies,antibody fragments, antigens, hormone receptors, parasites, cells,haptens, metabolites, allergens, nucleic acids, nuclear materials,autoantibodies, blood proteins, cellular debris, enzymes, tissueproteins, enzyme substrates, coenzymes, neuron transmitters, viruses,viral particles, microorganisms, proteins, polysaccharides, chelators,drugs, aptamers, peptides, and any other member of a specific bindingpair. This list only incorporates some of the many different materialsthat can be coated onto the substrate surface to produce a thin filmassay system. Whatever the selected analyte of interest is, thereceptive material is designed to bind specifically with the analyte ofinterest.

The blocking material may be any material that specifically does notbind the analyte of interest. The blocking material may be a passivematerial, such as milk proteins such as beta casein, albumin such asbovine serum albumin, and other proteins that do not recognize thetarget analyte; polymers such as polyvinyl pyrolidone, polyethyleneglycol, and/or polyvinyl alcohol; surfactants (e.g., Pluronics),carbohydrates, antibodies, DNA, PNA, ubiquitin, and streptavidin. Inother embodiments, the blocking material may be a receptive materialthat binds only with analytes different from the analyte of interest.

The matrix or medium containing the analyte of interest may be a liquid,a solid, or a gas, and can include a bodily fluid such as mucous,saliva, urine, fecal material, tissue, marrow, cerebral spinal fluid,serum, plasma, whole blood, sputum, buffered solutions, extractedsolutions, semen, vaginal secretions, pericardial, gastric, peritoneal,pleural, or other washes and the like. The analyte of interest may be anantigen, an antibody, an enzyme, a DNA fragment, an intact gene, a RNAfragment, a small molecule, a metal, a toxin, an environmental agent, anucleic acid, a cytoplasm component, pili or flagella component,protein, polysaccharide, drug, or any other material. For example,receptive material for bacteria may specifically bind a surface membranecomponent, protein or lipid, a polysaccharide, a nucleic acid, or anenzyme. The analyte which is specific to the bacteria may be apolysaccharide, an enzyme, a nucleic acid, a membrane component, or anantibody produced by the host in response to the bacteria. The presenceor absence of the analyte may indicate an infectious disease (bacterialor viral), cancer or other metabolic disorder or condition. The presenceor absence of the analyte may be an indication of food poisoning orother toxic exposure. The analyte may indicate drug abuse or may monitorlevels of therapeutic agents.

One of the most commonly encountered assay protocols for which thistechnology can be utilized is an immunoassay. However, the generalconsiderations apply to nucleic acid probes, enzyme/substrate, and otherligand/receptor assay formats. For immunoassays, an antibody may serveas the receptive material or it may be the analyte of interest. Thereceptive material, for example an antibody or an antigen, should form astable, reactive layer on the substrate surface of the test device. Ifan antigen is to be detected and an antibody is the receptive material,the antibody must be specific to the antigen of interest; and theantibody (receptive material) must bind the antigen (analyte) withsufficient avidity that the antigen is retained at the test surface. Insome cases, the analyte may not simply bind the receptive material, butmay cause a detectable modification of the receptive material to occur.This interaction could cause an increase in mass at the test surface, adecrease in the amount of receptive material on the test surface, or achange in refractive index. An example of the latter is the interactionof a degradative enzyme or material with a specific, immobilizedsubstrate. In this case, one would see a diffraction pattern beforeinteraction with the analyte of interest, but the diffraction patternwould disappear if the analyte were present. The specific mechanismthrough which binding, hybridization, or interaction of the analyte withthe receptive material occurs is not important to this invention, butmay impact the reaction conditions used in the final assay protocol.

In addition to producing a simple diffraction image, patterns ofanalytes can be such as to allow for the development of a holographicsensing image and/or a change in visible color. Thus, the appearance ofa hologram or a change in an existing hologram will indicate a positiveresponse. The pattern made by the diffraction of the transmitted lightcan be any shape including, but not limited to, the transformation of apattern from one pattern to another upon binding of the analyte to thereceptive material. In particularly preferred embodiments, thediffraction pattern becomes discernible in less than one hour aftercontact of the analyte with the biosensing device of the presentinvention.

The diffraction grating which produces the diffraction of light uponinteraction with the analyte must have a minimum periodicity of about ½the wavelength and a refractive index different from that of thesurrounding medium. Very small analytes, such as viruses or molecules,can be detected indirectly by using a larger, “diffraction-enhancingelement,” such as a micro-particle, that is specific for the smallanalyte. One embodiment in which the small analyte can be detectedcomprises coating the enhancing particle, such as a latex bead orpolystyrene bead, with a receptive material, such as an antibody, thatspecifically binds to the analyte of interest. Particles that can beused in the present invention include, but are not limited to, glass,cellulose, synthetic polymers or plastics, latex, polystyrene,polycarbonate, proteins, bacterial or fungal cells, silica, celluloseacetate, carbon, and the like. The particles are desirably spherical inshape, but the structural and spatial configuration of the particles isnot critical to the present invention. For instance, the particles couldbe slivers, ellipsoids, cubes, random shape and the like. A desirableparticle size ranges from a diameter of approximately 0.1 micron to 50microns, desirably between approximately 0.1 micron and 2.0 microns. Thecomposition of the particle is not critical to the present invention.

Desirably, the receptive material layer on the substrate willspecifically bind to an epitope on the analyte that is different fromthe epitope used in the binding to the enhancing particle. Thus, fordetecting a small analyte in a medium, the medium is first exposed tothe latex particles having the virus-specific receptive materialthereon. The small analytes of interest in the medium will bind to thelatex particles. Then, the latex particles are optionally washed andexposed to the biosensor film with the pattern of active receptivematerial areas containing the virus-specific antibodies. The antibodiesthen bind to the viral particles on the latex bead thereby immobilizingthe latex beads in the same pattern as the active areas on the film.Because the bound latex beads will cause diffraction of the visiblelight, a diffraction pattern is formed, indicating the presence of theviral particle in the liquid. Other combinations using diffractionenhancing particles are described, for example, in U.S. Pat. No.6,221,579 incorporated herein for all purposes.

Any one of a wide variety of materials may serve as the substrate towhich the receptive material and blocking material are applied. Suchmaterials are well known to those skilled in the art. For example, thesubstrate may be formed of any one of a number of suitable plastics,metal coated plastics and glass, functionalized plastics and glass,silicon wafers, glass, foils, etc. Rather than requiring a rigidsubstrate for the photopatterning process described herein, it has beenfound that thermoplastic films are quite suitable. Such films include,but are not limited to, polymers such as: polyethylene-terephthalate(MYLAR®), acrylonitrile-butadiene-styrene, acrylonitrile-methyl acrylatecopolymer, cellophane, cellulosic polymers such as ethyl cellulose,cellulose acetate, cellulose acetate butyrate, cellulose propionate,cellulose triacetate, cellulose triacetate, polyethylene,polyethylene-vinyl acetate copolymers, ionomers (ethylene polymers)polyethylene-nylon copolymers, polypropylene, methyl pentene polymers,polyvinyl fluoride, and aromatic polysulfones. Preferably, the plasticfilm has an optical transparency of greater than 80 percent. Othersuitable thermoplastics and suppliers may be found, for example, inreference works such as the Modern Plastics Encyclopedia (McGraw-HillPublishing Co., New York 1923-1996).

In one embodiment of the invention, the thermoplastic film may have ametal coating. The film with metal coating thereon may have an opticaltransparency of between approximately 5 percent and 95 percent. A moredesired optical transparency for the thermoplastic film used in thepresent invention is between approximately 20 percent and 80 percent. Ina desired embodiment of the present invention, the thermoplastic filmhas at least an approximately 80 percent optical transparency, and thethickness of the metal coating is such as to maintain an opticaltransparency greater than about 20 percent, so that diffraction patternscan be produced by either reflected or transmitted light. Thiscorresponds to a metal coating thickness of about 20 nanometers.However, in other embodiments of the invention, the metal thickness maybe between approximately 1 nanometer and 1000 nanometers.

The preferred metal for deposition on the film is gold. However, silver,aluminum, chromium, copper, iron, zirconium, platinum, nickel, andtitanium, as well as oxides of these metals, may be used. Chromium oxidecan be used to make metalized layers.

The receptive material and blocking material may be applied to thesubstrate over the photo-reactive crosslinking agent by any conventionalmethod. The material is applied so that it generally uniformly covers anentire (for example, upper) surface of the substrate. Non-contactmethods for applying the materials may be desired so as to eliminate thepossibility of contamination by contact during application. Suitableapplication methods include, but are not limited to, dipping, spraying,rolling, spin coating, and any other technique wherein the receptivematerial layer can be applied generally uniformly over the entire testsurface of the substrate. Simple physisorption can occur on manymaterials, such as polystyrene, glass, nylon, metals, polycarbonate, orother materials well known to those skilled in the art. One particularembodiment of immobilizing the analyte-specific receptive material layerinvolves molecular attachment, such as that possible between thiol ordisulfide-containing compounds and gold. Typically, a gold coating ofabout 5 to about 2000 nanometers thick is supported on a silicon wafer,glass, or polymer film (such as a MYLAR® film). The analyte-specificreceptor attaches to the gold surface upon exposure of a solution of thereceptive material.

Although not preferred, the invention also includes contact printingmethods of applying the materials. The technique selected shouldminimize the amount of receptive material required for coating a largenumber of test surfaces and maintain the stability/functionality of thereceptive material during application. The technique should also applyor adhere the receptive material to the substrate in a uniform andreproducible fashion.

It is also contemplated that the receptive material layer may be formedon the substrate as a self-assembling monolayers of alkanethiolates,carboxylic acids, hydroxamic acids, and phosphonic acids on metalizedthermoplastic films. The self-assembling monolayers have receptivematerial bound thereto. Reference is made to U.S. Pat. No. 5,922,550 fora more detailed description of such self-assembling monolayers andmethods for producing the monolayers. The '550 patent is incorporatedherein in its entirety for all purposes.

The mask may be formed of any suitable material that shields theunderlying portion of the substrate from the irradiating energy source.A material that has proven useful for defining patterns of active andinactive receptive material regions on a gold-plated MYLAR® film coatedwith an antibody solution is a transparent or translucent polymer film(such as MYLAR®) having a pattern of shielded regions printed thereon.This type of mask is useful for light sources (irradiating energysource) with a wavelength to greater than or equal to about 330nanometers. For light sources having a wavelength below about 330nanometers, a quartz or fused silica mask having chrome or other metalplated blocked regions defined thereon may be used. It may be desired toselect a hole pattern and size so as to maximize the visible diffractioncontrast between the active and inactive regions. It has been foundsuitable if the active regions are defined as generally circular with adiameter of about 10 microns and spaced from each other by about 5microns.

Any suitable energy source may be selected for irradiating the mask andsubstrate combination. An energy source is selected particularly foractivating the specific type of photo-reactive crosslinking agent. Theenergy source may be, for example, a light source, e.g., an ultraviolet(UV) light source, an electron beam, a radiation source, etc. In oneparticular embodiment, the photo-reactive crosslinking agent is SANPAHand the activating energy source is a UV light source. The sensor isexposed to the light source for a period of time sufficient for thephoto-reactive group of the crosslinking agent to be activated and thuslink or bind with either the receptive material or blocking material. Itshould be appreciated that the invention is not limited to anyparticular type of light or activating energy source or exposure times.The type of light (e.g., wavelength) and exposure times may varydepending on the particular type of photo-reactive crosslinking agent.Other suitable energy sources may include tuned lasers, electron beams,various types of radiation beams including gamma and X-ray sources,various intensities and wavelengths of light including light beams ofsufficient magnitude at the microwave and below wavelengths, etc. Careshould be taken that the energy source does not damage (e.g., melt) theunderlying substrate or mask.

FIG. 1 is a schematic representation of one method for producingbiosensors according to the invention. In this example, the receptivematerial is applied first prior to the masking process such that thepattern of receptive material areas corresponds to the pattern ofexposed areas in the mask. It should also be understood that the stepsare carried out in a controlled light protected environment.

Step A represents the photo-reactive crosslinking agent 2 applied to asubstrate member 4. Excess agent may be rinsed or washed from thesubstrate.

Step B represents the receptive material (biomolecules) layer 6 appliedto the substrate member 4 over the crosslinking agent 2.

Step C depicts the mask 8 disposed over the substrate member 4. The mask8 includes exposed or open regions 10 and shielded regions 12 definedthereon.

Step D represents the mask 8 and substrate member 4 combination beingirradiated with an energy source 14. It can be seen that the areas ofthe substrate member 4 underlying the shielded regions 12 of the mask 8are protected from the energy source 14. The photo-reactive groups ofthe crosslinking agent 2 exposed to the energy source 14 through theopen regions 10 of the mask 8 are activated by the energy source 14 andlink with the biomolecules 6 in the exposed areas. The crosslinkingagent 2 underlying the shielded regions 12 of the mask 8 is not exposedto the energy source and is thus unactivated. The biomolecules 6 inthese regions are thus not linked or attached with the agent 2 and areremoved in a subsequent cleaning (i.e., rinsing) step.

Step E represents the biosensor after the masking process and removal ofthe unreacted receptive material.

Step F represents the biosensor after a layer of blocking material 16has been applied to the substrate member 4. The FIGURE depicts theblocking material as separate molecules dispersed between the attachedbiomolecules 6. However, it should be appreciated that this is forillustrative purposes only. The blocking material would likely beapplied as a uniform coating over the entire substrate member.

Step G represents the substrate member being irradiated the second timewith the energy source 14 to activate the remaining crosslinking agent 2in the areas previously shielded by the mask 8. The photo-reactivegroups of the crosslinking agent 2 are activated and link with or attachthe blocking molecules 16.

Step H represents the biosensor in its final form after cleaning orrinsing of any excess blocking material 16. The biosensor includes apattern of active areas of the receptive material 6 corresponding to theexposed areas of the mask 8, and a pattern of areas of the blockingmaterial 16 corresponding to the shielded areas of the mask 8.

The biosensors according to the invention have a wide range of uses inany number of fields. The uses for the biosensors of the presentinvention include, but are not limited to, detection of chemical orbiological contamination in garments, such as diapers, generally thedetection of contamination by microorganisms in prepacked foods such asmeats, fruit juices or other beverages, and the use of the biosensors ofthe present invention in health diagnostic applications such asdiagnostic kits for the detection of proteins, hormones, antigens,nucleic acids, DNA, microorganisms, and blood constituents. The presentinvention can also be used on contact lenses, eyeglasses, window panes,pharmaceutical vials, solvent containers, water bottles, band-aids,wipes, and the like to detect contamination. In one embodiment, thepresent invention is contemplated in a dipstick form in which thepatterned substrate is mounted at the end of the dipstick. In use thedipstick is dipped into the liquid in which the suspected analyte may bepresent and allowed to remain for several minutes. The dipstick is thenremoved and then, either a light is projected through the substrate orthe substrate is observed with a light reflected from the substrate. Ifa diffraction pattern is observed, then the analyte is present in theliquid.

In another embodiment of the present invention, a multiple analyte testis constructed on the same support. A strip may be provided with severalpatterned substrate sections. Each section has a different receptivematerial that is different for different analytes. It can be seen thatthe present invention can be formatted in any array with a variety ofpatterned substrates thereby allowing the user of the biosensor deviceof the present invention to detect the presence of multiple analytes ina medium using a single test.

In yet another embodiment of the present invention, the biosensor can beattached to an adhesively backed sticker or decal which can then beplaced on a hard surface or container wall. The biosensor can be placedon the inside surface of a container such as a food package or a glassvial. The biosensor can then be visualized to determine whether there ismicrobial contamination.

It should be understood that the present invention includes variousother embodiments, modifications, and equivalents to the examples of theinvention described herein which, after reading the description of theinvention, may become apparent to those skilled in the art withoutdeparting from the scope and spirit of the present invention.

1. A method of making a biosensor, comprising the steps of: applying aphoto-reactive crosslinking agent directly to a surface of a substratemember; forming one of a receptive material layer and a blockingmaterial layer over the crosslinking agent; placing a mask over thesubstrate member, the mask having a configuration so as to shield atleast one underlying area of the substrate member while exposing atleast one adjacent area, and irradiating the substrate member and maskcombination with an energy source sufficient to activate thecrosslinking agent in the areas exposed by the mask, the activatedcrosslinking agent crosslinking with the respective receptive materialor blocking material in the exposed areas; cleaning the unreactedreceptive material or blocking material from the shielded areas of thesubstrate member after removal of the mask; forming a layer of therespective other of the blocking material and receptive material overthe surface of the substrate member, and irradiating the substratemember with the energy source so as to activate the remainingcrosslinking agent in the previously shielded areas, the activatedcrosslinking agent crosslinking with the respective blocking material orreceptive material; and wherein a resulting pattern of active receptivematerial areas and blocking material areas are defined according to thepattern of shielded and exposed areas of the mask.
 2. The method as inclaim 1, comprising forming the receptive material layer on thesubstrate before the blocking material layer such that the pattern ofactive areas of receptive material correspond to the exposed areas ofthe mask.
 3. The method as in claim 1, comprising selecting thesubstrate member from the group of materials consisting of plastics,metal coated plastics and glass, functionalized plastics and glass,silicon wafers, glass, and foils.
 4. The method as in claim 1, whereinthe substrate member comprises a polymer film coated with a metal. 5.The method as in claim 4, comprising selecting the metal from the groupconsisting of gold, silver, chromium, nickel, platinum, aluminum, iron,copper, gold oxide, chromium oxide, titanium, titanium oxide, silicone,silicone oxide, silicone nitride, silver oxide, or zirconium.
 6. Themethod as in claim 1, wherein the receptive material in the active areasfacilitates diffraction of transmitted or reflected light in adiffraction pattern corresponding to the active receptive materialareas, and further comprising viewing the diffraction pattern of activeareas of receptive material with the naked eye.
 7. The method as inclaim 1, wherein the receptive material is protein based.
 8. The methodas in claim 6, wherein the receptive material is an antibody.
 9. Themethod as in claim 1, comprising irradiating the substrate member withUV light at a wavelength sufficient for activating the photo-reactivecrosslinking agent exposed through the mask.
 10. The method as in claim1, comprising selecting the receptive material from at least one ofantigens, antibodies, nucleotides, chelators, enzymes, bacteria, yeasts,fungi, viruses, bacterial pill, bacterial flagellar materials, nucleicacids, polysaccharides, lipids, proteins, carbohydrates, metals,hormones, peptides, aptamers and respective receptors for saidmaterials.
 11. The method as in claim 1, wherein the analyte of interestis selected from at least one of a bacteria, yeast, fungus, virus,rheumatoid factor, IgG, IgM, IgA, IgD and IgE antibodies,carcinoembryonic antigen, streptococcus Group A antigen, viral antigens,antigens associated with autoimmune disease, allergens, tumor antigens,streptococcus group B antigen, HIV I or HIV II antigen, antibodiesviruses, antigens specific to RSV, an antibody, antigen, enzyme,hormone, polysaccharide, protein, lipid, carbohydrate, drug, nucleicacid, Neisseria meningitides groups A, B, C, Y and W sub 135,Streptococcus pneumoniae, E. coli K1, Haemophilus influenza type A/B, anantigen derived from microorganisms, PSA and CRP antigens, a hapten, adrug of abuse, a therapeutic drug, an environmental agents, or antigensspecific to Hepatitis.
 12. The method as in claim 1, wherein thephoto-reactive crosslinking agent is one of SANPAH (N-Succinimidyl2-[p-azido-salicylamido]ethyl-1,3′-dithiiopropionate); SAND(Sulfosuccinimidyl2-[m-azido-o-nitro-benzamido]ethyl-1,3′-dithiopropionate); and ANB-NOS(N-5-Azido-2-nitrobenzoyloxysuccinimide).